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Department of Biology, Sections of General Physiology and Comparative Anatomy, University of Ferrara, 44100-I Ferrara, Italy
Address all correspondence and requests for reprints to: Dr. Maria E. Ferretti, Department of Biology, Section of General Physiology, University of Ferrara, via Luigi Borsari 46, 44100-I Ferrara, Italy. E-mail: clm{at}dns.unife.it.
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Abstract |
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. RT-PCR evidences the presence of mRNA for ERß, not for ER. Our data suggest that 17ß-E2 stimulates [3H]AA release from WISH cells through an apparently nongenomic pathway and interaction with membrane binding sites. These last are, at least in part, similar if not identical to ERß. |
Introduction |
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Fetal membranes of several species, human included, are able to synthesize estrogens (1, 2, 6). Indeed, particularly high hormone levels have been found in human chorion and decidua, and they significantly increase after spontaneous vaginal delivery (7). Moreover, an increase of the estrogen/progesterone ratio is observed in the amniotic fluid of women in active labor compared with those not in labor (8). Fetal membranes represent not only a site of estrogen synthesis and release but also a target for such hormones. In fact, ER mRNAs have been found in human amnion and chorion, and it has been shown that their concentrations in chorio-decidua are 3-fold higher in tissues obtained after spontaneous labor onset than in tissues obtained from cesarean section at a similar gestational age but before labor onset (9). Moreover, it has been demonstrated recently that both ER subtypes, ER and ERß, are expressed in rhesus monkey amnion and chorio-decidua (10).
Estrogen actions at the level of fetal membranes also contribute to preparing the uterus for delivery; in fact, it has been demonstrated that these hormones stimulate oxytocin gene expression in human chorio-decidua (9) and PG release from human amniotic cells obtained after spontaneous labor and vaginal delivery (5), as well as from amnion-like cells (11).
For several years, we have addressed the mechanisms responsible for the onset of labor, and in particular the regulation of PG release from fetal membranes, using the human amnion-like Wistar Institute Susan Hayflick (WISH) cells, which are considered a good model for analysis of the physiological functions of amnion cells as well as for the characterization of modulation by different agonists of PGE2 release. As a matter of fact, PGE2 has been found to be the main prostanoid produced by both amniocytes and WISH cells; moreover, PGE2 output from WISH cells is evoked by the same classes of agonists (11, 12, 13, 14, 15) that are effective in amnion cells (5, 16, 17, 18, 19).
We have recently demonstrated that 17ß-estradiol (17ß-E2) dose-dependently inhibits PGE2 release from WISH cells pretreated with the cAMP elevating agent Ro 20-1724 but stimulates PGE2 output from cells unexposed to the drug. In both conditions, the hormone effect is counteracted by the ER antagonist tamoxifen, by the protein synthesis inhibitor cycloheximide, or when 17ß-E2 is administered together with BSA (11), which could impair the steroid diffusion through the cell membrane. Moreover, by means of the classical binding techniques, we have evidenced the presence of specific receptors for 17ß-E2 in whole WISH cells, but only when they are pretreated with Ro 20-1724 or forskolin (11). On the basis of these observations, we have hypothesized that WISH cells possess few constitutive 17ß-E2 receptors, whose activation leads to an enhancement of PGE2 release; when exposed to cAMP-elevating agents, new receptors predisposed to inhibit prostanoid output become available.
As a continuation of our research, we investigated a possible influence of 17ß-E2 on the release of the PGE2 precursor, arachidonic acid (AA), from WISH cell membrane phospholipids. Moreover, because our previous studies demonstrated the presence of ERs in WISH cells but provided no information about their characterization and distribution in the cell, we analyzed the above issues by means of RT-PCR techniques as well as fluorescence labeling assays.
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Materials and Methods |
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-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]-estra-1,3,5 (10)-triene-3,17ß-diol (ICI 182,780) was obtained from Tocris Cookson Ltd. (Bristol, UK). The antibody H-150 was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), the antibody ER-21 was the kind gift of G. Greene (Chicago, IL), the FITC-labeled goat antirabbit IgG serum was purchased from Vector Laboratories (Burlingame, CA). Tissue culture media, sera, and RT-PCR reagents were purchased from Invitrogen (Paisley, Scotland, UK). All other chemicals were the highest reagent grades commercially available.
Cell culture
Amnion-like WISH cells (ATCC CCL-25; American Type Culture Collection, Manassas, VA) were grown at 37 C in an atmosphere of 5% CO2/95% air, in a mixture of Ham’s F12 and DMEM (1:1 vol/vol) supplemented with 10% fetal bovine serum, 30 µg/ml gentamicin and 0.25 µg/ml amphotericin B. The cells were seeded into 24-well plates at 2 x 105 cells per well in F12/DMEM + 10% fetal bovine serum, and grown to about 70% confluence (2–3 d).
Incorporation and release of [3H]AA
Radiolabelling of the cells (2 x 105 cells per well) with [3H]AA was achieved by including 0.5 µCi/well in the serum-free medium 18 h before assay, because radioactivity incorporated by WISH cells was maximal at this time. Cells were then washed three times with pseudoamniotic fluid (PAF) containing 118.5 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.15 mM KH2PO4, 1.15 mM MgSO4, and 25.0 mM NaHCO3, supplemented with 2.0 mM glucose, 6.0 mM urea, and 0.2% BSA (pH 7.0), previously gassed with a 95% O2/5% CO2 mixture. Cells were supplied with PAF at a constant flow rate of 0.3 ml/min by a four-channel peristaltic pump (Gilson, Villier Le Bel, France), then perifused with PAF for 1 h before treatment to obtain a stable [3H] AA basal release. The test substances were infused into the wells by means of the same pump. Fractions of perifusate were collected every 3 min, and radioactivity of the perifusate solution was determined by means of a Beckman LS 6500 scintillation spectrometer. In some experiments, BSA-free PAF was used for cell washing and perifusion. [3H] AA release was measured as dpm/3-min fraction. Basal release ranged from 150–200 dpm/3-min fraction among the different cell cultures, and it remained substantially unchanged throughout the entire experiment, which lasted up to 2.5 h. Experiments were performed at least in triplicate, using different cell cultures.
The test substances were dissolved in ethanol (17ß-E2, Ro 20-1724, AACOCF3, 10-2 M), in DMSO (ICI 182,780 and cycloheximide, 10-2 M and 5 mg/ml, respectively), or in PAF (E2coBSA, 10-5 M). When requested, they were then diluted with PAF plus BSA or BSA-free PAF and added to the perifusion system. Ethanol and DMSO, at the doses used, did not interfere with the assay performed.
PGE2 level determination
For PGE2 level determination, the medium was removed from cells and replaced with fresh serum-free F12/DMEM containing test substances. After incubation of samples for 30 min, the media were collected and stored at -80 C. The amount of PGE2 was assayed in the collected media by an RIA procedure, as previously described (11). Data are expressed as nanograms of PGE2 produced per 106 cells. Experiments were performed in triplicate, using different cell cultures. Assay sensitivity was 40 pg/106 cells, and the intraassay or interassay coefficients of variations were less than 10%. 17ß-E2 was dissolved in ethanol (10-2 M) and E2coBSA in serum-free F12/DMEM (10-5 M); they were then diluted with serum-free F12/DMEM. Ethanol, at the dose used, did not interfere with the assay performed.
Labeling with E2coBSA-FITC
WISH cells were incubated in serum-free medium 24 h before the experiments; then cells (1.5 x 106/ml) were allowed to adhere onto glass coverslips overnight. Intact cells were washed twice with PBS+ solution [140 mM NaCl, 2.7 mM KCl, 6.4 mM Na2HPO4, 1.4 mM KH2PO4, 0.5 mM MgCl2, 0.9 mM CaCl2 (pH 7.2)] and incubated at 37 C for different times (1, 15, or 60 min) with E2coBSA-FITC at different concentrations (1.4 x 10-7, 1.4 x 10-6, and 1.4 x 10-5 M in PBS+). For competition studies, cells were preincubated with 10-5 M 17ß-E2 or the vehicle (0.1% ethanol in PBS+) for 5 min; this was followed by the addition of the different doses of E2coBSA-FITC and incubation for 15 min at 37 C. Other experiments were performed by adding 5% BSA in PBS+, 15 min before incubation with E2coBSA-FITC. Experiments were also made on cells permeabilized then labeled with E2coBSA-FITC. After incubation with E2coBSA-FITC, cells were washed with PBS (0.01 M; pH 7.2), postfixed with 1% paraformaldehyde (PFA) in PBS for 1 min and embedded in PBS/glycerol (1:1).
Localization of ER
Adherent WISH cells were prefixed with 0.5% PFA for 2 min, permeabilized, and labeled with different rabbit antibodies raised against the human ER (ER-21 and H-150; working dilutions in PBS, 5 and 2 µg/ml, respectively) for 1 h at room temperature. The antibodies (at the same dilution) were also used in prefixed cells without any permeabilization. Then, cells were washed in PBS and incubated with a FITC-labeled goat antirabbit IgG serum (working dilution, 1:100 in PBS) for 1 h at room temperature. Controls included labeling with secondary antibody alone, in the absence of primary antibody. The cells were postfixed with 3% PFA in PBS for 1 min and embedded in PBS/glycerol (1:1).
Cell permeabilization
Permeabilization was performed by incubating the cells for 2 min in the presence of PBS+ containing 0.05% Tween 20 and 0.5% BSA.
Confocal laser scanning microscopy
The laser scanning confocal microscope LSM 410 equipped with a Axiovert 100 TV microscope (Zeiss, Heidelberg, Germany) was used for the analysis, with FITC fluorescence excitation at 488 nm. Z-series optical sections, taken at 1-µm intervals, were evaluated using Adobe Photoshop 6.0 for Windows (Adobe Systems, Mountain View, CA).
RT-PCR
Amplification of individual RNA molecules was achieved by a method that combines RT and the PCR (RT-PCR). First, strand cDNA was synthesized using 5 µg total RNA, 3.5 µM oligo(dT)23, 500 µM 2'deoxynucleoside-5'triphosphates, and 2 U avian myeloblastosis virus reverse transcriptase. The samples were incubated at 37 C for 1 h in a reaction volume of 20 µl. PCR amplification from reverse transcribed cDNA was carried out using specifically designed PCR primers for ERß and ER (20) (ERß sense, 5'-TGAAAAGGAAGGTTAGTGGGAACC-3'; ERß antisense, 5'-TTGTCAGGGACATCATCATGG-3'; ER sense, 5'-GTGCCTGGCT AGAGATCCTG-3'; ER antisense, 5'-TTGTGCATGATGAGGGTAAA-3'). PCR amplification for glyceraldehyde phosphodehydrogenase (GAPDH) was performed on the same samples as a parallel control (GAPDH sense, 5'-CCACCCATGGCAAATTCCATGGCA-3'; GAPDH antisense, 5'-TCTAGACGGCAGGTCAGGTCCACC-3') (21). A 1/20 volume of the generated cDNA reaction was used in the amplification reaction. PCR was performed in a 25-µl volume using 1.5 mM MgCl2, 0.2 mM 2'deoxynucleoside-5'triphosphates, 0.25 µg of each sense and antisense primer, and 0.25 U of Taq polymerase. PCR conditions consisted of 35 cycles of amplification, using the following parameters: denaturation at 94 C for 30 sec; annealing at 50 C (ERß), 48 C (ER), or 55 C (GAPDH) for 30 sec; and extension at 72 C for 30 sec. The amplified products were separated on a 1.5% agarose gel containing ethidium bromide, using a DNA ladder as size marker.
Statistical analysis
Statistical significance of data was assessed by ANOVA, followed by Dunnett’s or Bonferroni’s post test. When two populations of unpaired data were compared, one-tail t test was used. The computer program PRISM (version 3.0, Graph Pad Inc., San Diego, CA) was used.
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Results |
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) or presence of the cAMP phosphodiesterase inhibitor Ro 20-1724 at 10-5 M (Fig. 1B). As shown, similar actions were obtained in both conditions: the hormone dose-dependently stimulated [3H]AA release, evoking a statistically significant (P < 0.05) output at 10-8 M and the maximal stimulation (about 5.5-fold, peak value, vs. basal level) at the highest dose tested (10-6 M). In subsequent experiments, only untreated cells were used, and the hormone concentration chosen was 10-6 M.
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illustrates the effect of the ER antagonist, ICI 182,780 (10-5 M), on basal as well as 17ß-E2-stimulated [3H]AA release. This drug was without effect in basal conditions, but significantly (P < 0.01) reduced the steroid-evoked release.
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). Otherwise, the effect of 17ß-E2 remained substantially unchanged in WISH cells pretreated for 30 min with cycloheximide (5 µg/ml) (Fig. 4), a protein synthesis inhibitor that influenced neither cell viability, as determined by the trypan blue exclusion test, nor [3H]AA release in the absence of the hormone.
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, basal as well as hormone-stimulated [3H]AA releases were very similar to those measured in cells perifused with PAF plus BSA; this protein (0.2%), as expected, significantly (P < 0.05) enhanced [3H]AA output (about 2.5-fold, peak value vs. basal level), and the combination BSA plus 17ß-E2 evoked a stimulation significantly higher (P < 0.05) than that observed in the presence of BSA alone. The effect of both 17ß-E2 and BSA was maintained in WISH cells pretreated for 30 min with cycloheximide (5 µg/ml), then perifused with BSA-free PAF (Fig. 6). The stimulatory action of 17ß-E2 on [3H]AA release from WISH cells perifused with BSA-free PAF was again reduced by ICI 182,780 and unaffected by cell preincubation in the presence of Ro 20-1724 (data not shown).
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).
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, the drug (10-5 M) significantly (P < 0.01) stimulated [3H]AA release (about 3-fold, peak value vs. basal level) from WISH cells perifused with BSA-free PAF, even if at a lesser extent (P < 0.05) with respect to free 17ß-E2 (10-6 M).
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, the effects of 17ß-E2 or E2coBSA on PGE2 output from WISH cells are reported. As shown, the free steroid (10-9 to 10-5 M) dose-dependently stimulated the prostanoid release, reaching a maximum (180%) at 10-6 M. On the contrary, E2coBSA was completely ineffective at concentrations ranging from 10-9 to 10-5 M.
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In Fig. 9 the effect of 1.4 x 10-5 M E2coBSA-FITC is reported. As shown, the fluorescence was almost exclusively localized on the cell surface and increased progressively after incubation for 1, 15, and 60 min (Fig. 9, A–D). The binding was unaffected by excess free BSA (data not shown) and displaced by unlabeled 17ß-E2 (Fig. 9, E and F). Identically prepared cells, permeabilized as described in Materials and Methods, demonstrated intracellular (mainly cytoplasmic) labeling with E2coBSA-FITC (Fig. 10, A and B). H-150 (2 µg/ml), a rabbit polyclonal antiserum against a recombinant protein corresponding to the amino acids 1–150 mapping at the N terminus of human ERß, immunostained the outer cell membrane of a minority subset of intact WISH cells (<5%) (Fig. 10D). Cell permeabilization resulted in a dense intracellular (mainly in the peripheral cytoplasm) staining (Fig. 10C); in no instance was labeling seen within the nucleus. Otherwise ER-21 (5 µg/ml), a rabbit IgG affinity-purified peptide antibody raised to the first 21 amino acids mapping the N terminus of human ER, slightly labeled only very scarce cells (Fig. 10, E and F).
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and ERß. Using different primers of the carboxy terminus and the DNA-binding domain of ERß, RT-PCR revealed the same band corresponding to ERß (528 bp) previously detected by Lau et al. (20) (Fig. 11); on the contrary, ER mRNA was not detected in the same cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this paper, we report that 17ß-E2 greatly and dose-dependently enhances [3H]AA release from the human amnion-like WISH cells, through an apparently nongenomic action involving the hormone interaction with a membrane binding site. As a matter of fact, 1) 17ß-E2 action is fast and short-lived, being measurable 3 min after application and rapidly declining when hormone administration still persists; 2) [3H]AA release from membrane phospholipids occurs through the phospholipase A2 pathway; 3) 17ß-E2 action is unaffected by inhibition of protein synthesis; 4) the plasma membrane-impermeable E2coBSA also induces a significant increase of [3H]AA release from WISH cells; this effect cannot be exerted by the BSA moiety of the complex, due to very low concentration (0.066%). On the contrary E2coBSA is completely ineffective on PGE2 release, a response that is significantly enhanced by free 17ß-E2. This observation confirms our previous results indicating that an interaction of 17ß-E2 with intracellular receptors was needed to induce PGE2 release (11); moreover, it allows us to exclude the possibility that a significant amount of estradiol dissociates from E2coBSA.
In our experiments, E2coBSA is less effective than 17ß-E2 in inducing the cellular response. For this purpose, conflicting results have been reported in the literature: estradiol-BSA conjugates are as effective as 17ß-E2 in human vascular endothelial cells (28), but less effective in Chinese hamster ovary transfected cells expressing ER or ERß (26); the lower effectiveness of estradiol-BSA conjugates may be due to the BSA protein physically hindering 17ß-E2 binding to its receptor. In addition, Stevis et al. (29), demonstrating that estradiol-BSA conjugates and estradiol evoke differential effects in a neuroblastoma cell line, suggested that membrane receptors are different from the classical intracellular ERs and are not recognized by the 17ß-E2-BSA conjugate. Therefore, the lower biological activity of estradiol-BSA conjugates, compared with 17ß-E2, may also be due to the presence of heterogeneous ERs on cell membrane.
BSA is a normal component of PAF that stimulates per se arachidonate output in different systems, WISH cells included. In our experimental conditions, basal as well as 17ß-E2-evoked [3H]AA releases are substantially identical in cells perifused with PAF plus BSA or BSA-free PAF. This observation suggests that the presence, in the perifusion medium, of BSA (which can reversibly bind to 17ß-E2) does not significantly impair the hormone interaction with the membrane binding sites as well as their activation; moreover, BSA does not interfere with 17ß-E2 action in WISH cells or alter the basal release. These results can be explained considering that, in our system: 1) the stimulatory effect of BSA progressively decreases upon a prolonged exposure, disappearing 24 min after application; and 2) WISH cells are perifused with PAF for 1 h, time at which the stimulatory effect of BSA is already completely lost, before fraction collection for the evaluation of basal or evoked [3H]AA release. A similar declining of BSA-evoked [3H]AA release has been observed in intact rat aorta, exposed for 20 min to the protein (23).
The exact mechanism underlying BSA effect remains unknown. Beck et al. (23) recently proposed that BSA action, in vascular smooth muscle and endothelial cells, can be explained by a high-affinity binding of the protein to AA and its extraction from the cell membrane; a role in this process could be exerted by specific albumin binding proteins which, indeed, have been identified on the cell surface (30, 31).
The presence of membrane interaction sites for 17ß-E2 in WISH cells is strongly supported by our observation that the impeded ligand E2coBSA-FITC labels the surface of a subset of intact cells. Labeling is specific, because it is unaffected by cell preincubation with an excess of free BSA but inhibited by previous exposure to 17ß-E2.
The E2coBSA-FITC compound has been shown to label a membrane ER in several cell types (24, 25, 26); in many instances only a low percentage of cells are labeled at their surface (26, 27, 28), as we observed in WISH cells. Fairly high concentrations of the fluorescent conjugate are needed to visualize binding on the surface of cells, probably for the same reasons invoked to justify the lower potency of E2coBSA, compared with 17ß-E2, in inducing cellular responses.
In addition to the plasma membrane, ERs have also been detected in the cytoplasm of WISH cells, as suggested by E2coBSA-FITC labeling of permeabilized cells. Intracellular labeling seems not to be the consequence of internalization processes observed in other cell types (32), because it is not significantly observed in intact cells even after long periods of incubation.
ERs identified in WISH cells, both on the membrane and inside of the cell, appear, at least in part, similar if not identical to ERß, as suggested by our demonstration that a specific antibody against this receptor subtype labels the surface of intact WISH cells or the intracellular space after permeabilization. Otherwise, ER21 antibody against a functional domain of the classical ER dealing with transcription regulation (33) fails to label intact as well as permeabilized WISH cells. These data are in line with our RT-PCR results, which demonstrate the presence, in WISH cells, of mRNA for ERß but not for ER.
Membrane ERs identical, or structurally related, to at least one form of the classical ER or ERß have been localized in several cells; meanwhile, evidence for a membrane ER unrelated to both subtypes has also been obtained (27, 34). Two findings [i.e. 1) ICI 182,780, a pure antagonist of the classical ERs (35), only partially inhibits the 17ß-E2-evoked [3H]AA release from WISH cells; and 2) the membrane labeling by the anti-ERß antibody is apparently seen in a lower number of intact WISH cells compared with cells labeled by E2coBSA-FITC] can lead to speculation that heterogeneous ERs are present on WISH cell membrane. However, further experiments are needed to support this preliminary suggestion.
In previous experiments, we have demonstrated that 17ß-E2 modulates PGE2 release from WISH cells through an action that involves an interaction with intracellular receptors and new protein synthesis (11). On the basis of these data and those here reported, we hypothesize that 17ß-E2 evokes different actions in WISH cells, through separate mechanisms: 1) interacting with a membrane binding site, the hormone induces a rapid arachidonate release from membrane phospholipids; and 2) interacting with an intracellular receptor, it modulates PGE2 output. The existence of separate action mechanisms for 17ß-E2 in WISH cells is further demonstrated by the observation that cell preincubation with the cAMP elevating agent, Ro 20-1724, does not influence the hormone-evoked [3H]AA release. Otherwise, 17ß-E2 significantly inhibits PGE2 release from cells preincubated with Ro 20-1724 but exerts an evident stimulatory effect on prostanoid output in untreated cells (11).
Provided that our in vitro results are applicable to the amnion tissue in vivo, it can be hypothesized that AA, rapidly released in the presence of 17ß-E2, may serve as the precursor for PGE2 synthesis, a process that is indeed stimulated by the steroid when intracellular cAMP levels are low. In this way, the two different modes of 17ß-E2 action can operate together, leading to the production of active prostanoids probably involved in labor initiation and maintenance. Alternatively, AA may act itself as a second messenger or be converted to eicosanoids different from PGE2; this second way seems obligatory when high intracellular cAMP levels are present, a condition in which 17ß-E2 indeed inhibits PGE2 release from amniotic cells (11).
In conclusion, through its action on AA metabolism in the amnion, 17ß-E2 could regulate still unidentified fetal membrane functions, or contribute to either enhancing or inhibiting uterine contractions. This divergent effect seems to be strictly dependent on intracellular cAMP levels that, in turn, are regulated by several agonists that target the amnion tissue, among which 17ß-E2 as well as PGs themselves (11, 36). The nucleotide could therefore exert a protective role on pregnancy maintenance not only directly, through its well-known relaxing effect on myometrium, but also indirectly inhibiting PGE2 synthesis by amnion in response to 17ß-E2 and possibly converting AA to eicosanoids able to reduce uterine contractions. Examples of hormonal regulation of amnionic AA metabolism, leading to the production of compounds that could inhibit myometrium activity, have already been described. To this purpose, Toth et al. (37) demonstrated that, in the amnion as well as in WISH cells, the human chorionic gonadotropin enhances the expression of both cyclooxygenase-1 and prostacyclin synthase, thus leading to the release of the tocolytic compound PGI2.
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Acknowledgments |
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Footnotes |
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Abbreviations: AA, Arachidonic acid; AACOCF3, 1,1,1-trifluoromethyl-6,9,12,15-heicosatetraen-2-one; DMSO, dimethylsulfoxide; 17ß-E2, 17ß-estradiol; E2coBSA, 17ß-estradiol 6-(O-carboxymethyl)oxime:BSA; E2coBSA-FITC, 17ß-estradiol 6-(O-carboxymethyl)oxime:BSA fluorescein isothiocyanate conjugate; ER, estrogen receptor; GADPH, glyceraldehyde phosphodehydrogenase; ICI 182,780, 7-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]-estra-1,3,5(10)-triene-3,17ß-diol; PG, prostaglandin; PAF, pseudoamniotic fluid; PFA, paraformaldehyde; WISH, Wistar Institute Susan Hayflick.
Received October 25, 2002.
Accepted for publication May 5, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxysteroid dehydrogenase activity supporting an increase in the estrogen/progesterone ratio of human fetal membranes at parturition. Am J Obstet Gynecol 168:1377–1385[Medline]
and ERß mRNA in intrauterine tissues of the pregnant rhesus monkey. Am J Physiol 278:C190–C198
in human amnionic WISH cells by various stimuli occurs through distinct intracellular mechanisms. J Pharmacol Exp Ther 280:1065–1074 and ER-ß in normal and malignant prostatic epithelial cells: regulation by methylation and involvement in growth regulation. Cancer Res 60:3175–3182 and ERß expressed in Chinese hamster ovary cells. Mol Endocrinol 13:307–319. Gynecol Obstet Invest 37:77–82[Medline]
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, P < 0.01 compared with 17ß-E2 alone (Bonferroni’s posttest).
